HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY (HPTLC) OF ECDYSTEROIDS PRESENT IN PLANT EXTRACTS COUPLED WITH IN SITU ANALYSIS AND IMAGING DESI/IMS/MS

Význam tématu

Ecdysteroids represent a class of polar, polyhydroxylated steroids originally characterized as insect moulting hormones but now recognized for their anabolic and pharmacological potential in human health. Reliable screening and identification of these compounds in complex plant extracts is critical for natural product discovery, quality control in nutritional supplements, and agricultural applications.

Cíle a přehled studie

This study aimed to develop and demonstrate a robust workflow combining high-performance thin-layer chromatography (HPTLC) with desorption electrospray ionization mass spectrometry imaging (DESI-MSI) and ion mobility separation (IMS) for the rapid screening, separation, and in situ characterization of ecdysteroids in plant extracts. Five methanolic extracts of Silene fimbriata and other related species were evaluated alongside standard ecdysteroids.

Použitá metodika a instrumentace

Chromatographic separation was performed on silica HPTLC plates using a chloroform/ethanol (4:1 v/v) mobile phase, with UV visualization at 254 nm prior to MS analysis. DESI-MS and DESI-IMS-MS experiments were conducted on a SYNAPT XS HDMS system equipped with a 2D DESI stage. Key parameters included positive ion mode, m/z range 100–1200, capillary voltage 4 kV, DESI solvent MeOH/H2O (98:2, v/v), and ion mobility wave velocities optimized for separation of isomeric ecdysteroids.

Hlavní výsledky a diskuse

Standard compounds produced distinct sodiated ions with minimal protonation or potassiation. Isobaric pairs such as ecdysone and ponasterone A were resolved by combined Rf and drift time (Dt) measurements (Rf 0.31/Dt 5.1 ms vs. Rf 0.55/Dt 5.37 ms). In plant extracts, the most intense TLC spot at Rf 0.28 corresponded to m/z 503.29, tentatively assigned to inokosterone or 20-hydroxyecdysone. Multiple IMS peaks for m/z 519.29 at Dt 4.7, 5.23, and 5.43 ms enabled differentiation of isomers and the assignment of polypodine B. Overall, DESI-MSI detected more analytes than UV imaging alone, and IMS added crucial orthogonal separation for structural isomer discrimination.

Přínosy a praktické využití metody

This integrated HPTLC–DESI-IMS–MSI approach offers:

• Rapid, multiplexed screening of crude and semi-purified plant extracts without extensive sample preparation
• Enhanced detection sensitivity beyond UV visualization, revealing low-abundance compounds
• Orthogonal separation via ion mobility to distinguish structural isomers and reduce false positives

These advantages support applications in phytochemical research, quality control of nutraceuticals, and discovery of bioactive natural products.

Budoucí trendy a možnosti využití

Future developments may include:

• Expansion of ion mobility databases for more accurate ecdysteroid identification
• Integration with high-throughput HPTLC platforms and automated image analysis
• Applicability to other complex natural product classes such as alkaloids or flavonoids
• Coupling with orthogonal MS/MS workflows for structural confirmation

Závěr

The demonstrated workflow combining HPTLC with DESI-IMS-MSI provides a powerful, flexible platform for the targeted screening and in situ analysis of ecdysteroids in plant extracts. It delivers superior detection, structural resolution, and rapid data acquisition, facilitating natural product research and quality assurance in industrial settings.

Reference

• Claude E., Towers M., Lafont R., Wilson I.D., Plumb R. High performance thin-layer chromatography of ecdysteroids present in plant extracts coupled with in situ analysis and imaging DESI/IMS/MS. Waters Corporation; 2021.
• Ecdybase Ecdysteroid Database.